P605: IMMUNOGENETICS AND ANTIGEN REACTIVITY PROFILING CONTRIBUTE TO UNRAVELLING THE ONTOGENY OF CLL STEREOTYPED SUBSET #4
Authors: Anastasia Iatrou, Electra Sofou, Eleni Kotroni, Lesley Ann Sutton, Michela Frenquelli, Raphael Sandaltzopoulos, Ioanna Sakellari, Niki Stavrogianni, Fotis Psomopoulos, Paolo Ghia, Richard Rosenquist, Andreas Agathangelidis, Anastasia Chatzidimitriou, Kostas Stamatopoulos
Journal: HemaSphere (2023)

Background: CLL subset #4 is the largest stereotyped subset in IGHV-mutated CLL (M-CLL). The clonotypic B cell receptor immunoglobulin (BcR IG) in subset #4, encoded by the IGHV4-34/IGKV3-20 gene pair, displays long heavy complementarity determining region 3 (VH CDR3), enriched in positively charged residues; ubiquitous expression of gamma heavy chain isotype; distinctive imprint of somatic hypermutation (SHM), characterized by the frequent introduction of acidic residues and pronounced intraclonal diversification. These features are reminiscent of edited autoantibodies, implicating ongoing (auto)antigen interactions in the natural history of CLL subset #4. Aims: Here, we sought to explore the immune trajectory and clonal dynamics of CLL subset #4, particularly focused on the role of SHM. Methods: We studied longitudinal samples from 6 subset #4 and 6 non-subset #4 IGHV4-34-expressing M-CLL cases. Clonotypic IG heavy (HC) and light chain (LC) gene rearrangements were profiled by next generation sequencing (NGS). The clonotype accounting for the majority of NGS reads in a given sample was defined as dominant; related clonotypes expressing the same IGHV/IGKV gene and VH CDR3 amino acid (aa) sequence yet differing in the aa sequence of the VH and/or VK domains were defined as subclonotypes. The dominant clonotypes of all cases as well as 32 subclonotypes of subset #4 cases were expressed as recombinant monoclonal antibodies (rmAbs) and screened for antigen reactivity using ELISA and flow cytometry. Results: NGS revealed two distinct patterns of subclonal architecture in the IGH repertoire of subset #4. The first was characterized by the presence of a single dominant clonotype (4 cases) whereas the second by the co-existence of ≥2 significantly expanded clonotypes with comparable frequencies (2 cases). Turning to the LC, 5/6 cases carried a single dominant clonotype, while 1 case carried ≥1 expanded clonotypes. In contrast, a single dominant clonotype was detected in all non-subset #4 cases for both HC and LC. Longitudinal analysis revealed significantly (p<0.05) more pronounced subclonal drift (changing relative frequencies) of the expanded clonotypes in subset #4 vs non-subset #4 cases, particularly for the HC. Surprisingly, in all subset #4 cases we identified clonotypic IgG transcripts (same IGH gene and VH CDR3 length, high VH CDR3 aa sequence identity) with 100% germline IGHV identity (‘truly unmutated’), present at low frequency (median: 0.003%, range: 0.0003-0.03%). Prompted by this, we produced rmAbs for both the dominant, somatically hypermutated BcR IG clonotype of all 6 subset #4 cases, as well as 32 truly unmutated subclonotypes (from all studied subset #4 cases) and compared their antigen reactivity profiles. We report consistent, significantly (p<0.05) stronger reactivity of the truly unmutated, subclonotypic BcR IG compared to the mutated BcR IG expressed by the dominant CLL #4 clone. With few exceptions, all subclonotypic truly unmutated BcR IG displayed more intense recognition against dsDNA and lipopolysaccharides, antigenic elements from CMV, Influenza A and Mycoplasma pneumoniae as well as viable HEK293 and Jurkat T cells. Summary/Conclusion: Our findings support that SHM in CLL subset #4 is functionally driven by persistent selection by (auto)antigens, ultimately leading to a significant redemption from autoreactivity. The identification of truly unmutated clonotypic IgG gene transcripts likely reflects a complex trajectory of clonal evolution, offering hints about the precise timing of SHM in relation to class switch recombination in the natural history of CLL subset #4. Keywords: Antigen presentation, B cell chronic lymphocytic leukemia, B cell lymphoma, Antibody